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During acute infectious mononucleosis (AIM), large clones of Epstein-Barr virus-specific T lymphocytes are produced. To investigate the dynamics of clonal expansion, we measured cell proliferation during AIM using deuterated glucose to label DNA of dividing cells in vivo, analyzing cells according to CD4, CD8 and CD45 phenotype. The proportion of labeled CD8(+)CD45R0(+) T lymphocytes was dramatically increased in AIM subjects compared to controls (mean 17.5 versus 2.8%/day; p<0.005), indicating very rapid proliferation. Labeling was also increased in CD4(+)CD45R0(+) cells (7.1 versus 2.1%/day; p<0.01), but less so in CD45RA(+) cells. Mathematical modeling, accounting for death of labeled cells and changing pool sizes, gave estimated proliferation rates in CD8(+)CD45R0(+) cells of 11-130% of cells proliferating per day (mean 47%/day), equivalent to a doubling time of 1.5 days and an appearance rate in blood of about 5 x 10(9) cells/day (versus 7 x 10(7) cells/day in controls). Very rapid death rates were also observed amongst labeled cells (range 28-124, mean 57%/day),indicating very short survival times in the circulation. Thus, we have shown direct evidence for massive proliferation of CD8(+)CD45R0(+) T lymphocytes in AIM and demonstrated that rapid cell division continues concurrently with greatly accelerated rates of cell disappearance.

CD45, a transmembrane protein tyrosine phosphatase (PTP), can either positively or negatively regulate Src-family protein tyrosine kinase (PTK) activity in vivo. It is proposed that TCR-initiated signaling requires the segregation of PTP activities from the engaged TCR, based upon the differential membrane compartmentalization on the T cell surface. To test the importance of CD45 exclusion from lipid microdomains for proper TCR signaling, a chimeric molecule was generated by fusing the CD45 cytoplasmic region, which contains the PTP domains, to the amino-terminal 12 amino acids of Lck, which target Lck to lipid microdomains. Using 3A9 T lymphocyte hybridoma (3A9H) cells whose TCR recognizes hen egg-white lysozyme (HEL), Lck-CD45 expression resulted in its targeting to lipid microdomains. The 3A9H cells expressing Lck-CD45 were reduced in their responses to HEL or co-cross-linking of CD3 and CD4, as assessed by IL-2 production and Ca(2+) mobilization. Src-family PTK activity associated with lipid microdomains was also decreased. These results suggest that the segregation of CD45 from proximal TCR signaling components is necessary for TCR signaling and that the targeting of CD45 PTP activity to lipid microdomains on the T cell surface results in decreased sensitivity of TCR-mediated signaling.

B cell receptor signaling threshold regulates negative selection of autoreactive B cells and determines the mechanism of B cell tolerance. Using mice carrying immunoglobulin transgene specific for MHC class I antigen K(k) (3-83 Tg mice), and IL-7-driven bone marrow (BM) culture system, we have previously shown that receptor editing is a major mechanism in B cell tolerance. To test the role of BCR signaling competence on the induction of tolerance-mediated receptor editing, we crossed the 3-83 Tg mice with mice deficient in CD45, a protein tyrosine phosphatase that functions asa positive regulator of the BCR signaling. We found that in the absence of self-antigen allelic exclusion is efficiently imposed in 3-83 Tg CD45(-/-) mice, although numbers of peripheral B cells are reduced. Using our BM culture system, we show here that immature 3-83 Tg CD45(-/-) B cells encountering self-antigen are developmentally arrested and undergo secondary light chain recombination and receptor editing, not different than CD45-sufficient cells. Thus, lack of CD45 does not abolish the receptor editing competence in immature B cells encountering high avidity membrane-bound antigen.

Anti-CD44 or anti-MHC II antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHC II, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHC II appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.

We investigated the protective functions of macrophages and gamma delta T cells in adult CD45 exon 6-deficient (CD45 -/-) mice against an intraperitoneal (i.p.) infection with Listeria monocytogenes. gamma delta T cells are preferentially localized in the spleen, liver, and intraperitoneal cavity of the adult CD45-/- mice. Increased numbers of gamma delta T cells were observed after i.p. infection with L. monocytogenes in the peritoneal cavity of C57BL/6 (CD45 +/+) mice but not in CD45 -/- mice. The gamma delta T cells showed predominant usage of V delta 5 and V delta 6 rearranged to J delta 1 in the infected CD45 -/- mice which are the same as those used by resident gamma delta T cells of noninfected CD45 +/+ and CD45 -/- mice. Furthermore, we analyzed the protective abilities of the CD45 -/-, CD45 +/+, and gamma delta T cell-depleted mice at the early stage of the listerial infection. The numbers of bacteria in the spleens and livers of the CD45 -/- mice 5 days after the listerial infection were almost ten times larger than those in the CD45 -/- and gamma delta T cell-depleted CD45 +/+ mice. Macrophages showed normal antigen presentation, nitric oxide production and bactericidal activity for L. monocytogenes despite their lacking CD45 surface expression, suggesting that CD45-negative macrophages have a minimal influence on the increased bacterial multiplication in the CD45-/- mice. These results suggest that the gamma delta T cells are induced by the bacterial infection in a CD45-dependent manner, and that unresponsiveness of the gamma delta T cells results in only weak protection against L. monocytogenes in CD45 -/- mice.

Lymphocytes circulating in the bloodstream home into lymph nodes (LN). T cells predominate in peripheral LN (PLN) and B cells in spleen or mucosal tissue, e.g. Peyer's patches (PP). DDD/1 mice are unique in marked paucity of LN cells, especially T cells. T cell frequency in PLN was 20-40% in this strain, compared to 60-80% in others. Immunohistochemistry confirmed the low density of T cells in the subcortical area but normal colonization of B cells in cortical area in PLN of DDD/1. In contrast, the T cell content of peripheral blood and spleen was higher in DDD/1 but that in PP was not significantly different compared to other strains. It was thus concluded that this abnormality in DDD/1 results from a homing defect of T cells into PLN but not from lymphopenia. Genetical analysis showed that the defect in T cell-specific homing was regulated by a single autosomal recessive gene, tentatively designated plt (paucity of lymph node T cells). Reciprocal bone marrow transplantation indicated that the plt phenotype may arise from some defect in PLN stroma but not in lymphocytes. An in vivo homing assay using fluorescence-labeled lymphocytes demonstrated that the homing defect was specific for T cells but not for B cells. A Stamper-Woodruff assay revealed that the binding between lymphocytes and PLN high endothelial venules was normal and that L-selectin and its ligand, peripheral node vascular addressin (PNAd), were expressed and functioned normally in DDD/1. These results taken together indicate that the T cell-specific homing into PLN is disturbed at a post-adhesion stage in DDD/1. The product of the plt locus may play a pivotal role at this stage.

Eight chemokines were tested for ability to elicit transendothelial chemotaxis of unstimulated peripheral blood T lymphocytes. The C-C chemokines monocyte chemotactic protein (MCP)-2, MCP-3, RANTES, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and, as previously described, MCP-1 induced significant, dose-dependent transendothelial chemotaxis of CD3+ T lymphocytes. In contrast, the C-X-C chemokines interleukin-8 (IL-8) and interferon-gamma inducible protein-10 (IP-10) failed to induce transendothelial chemotaxis of CD3+ T lymphocytes or T lymphocyte subsets. RANTES, MIP-1 alpha, and MIP-1 beta induced significant transendothelial chemotaxis of CD4+, CD8+, and CD45R0+ T lymphocyte subsets. Phenotyping of mononuclear cells that underwent transendothelial migration to MCP-2, MCP-3, RANTES, or MIP-1 alpha showed both monocytes and activated (CD26 high), memory-type (CD45R0+) T cells. Both CD4+ and CD8+ T lymphocytes were recruited, but not natural killer cells or significant numbers of B cells. MCP-2 was the only C-C chemokine tested that attracted a significant number of naive-type (CD45RA+) T lymphocytes. In the absence of endothelium, IL-8 but not IP-10 promoted modest but significant chemotoxis of CD3+ T lymphocytes. Our data support the hypothesis that C-C, not the C-X-C chemokines IL-8 or IP-10, promote transendothelial chemotaxis of T lymphocytes.

CD45 exon 6-deficient mice show a profound block of thymocyte development at a transitional differentiation step from immature CD4+ CD8+ to mature single-positive cells. Only a few T lymphocytes are observed in the periphery of such animals, but B cell development is not affected. We investigated whether mature B lymphocytes in CD45 exon 6-deficient mice have normal functions in the absence of CD45 expression and show here that CD45-defective B lymphocytes from CD45 exon 6-deficient mice have intact B lymphocyte functions, including T-dependent and T-independent antigen-specific antibody production and class switching in vivo if normal CD4+ T lymphocytes were adoptively transferred into the mutant mice. From these results, we conclude that CD45 expression on B lymphocytes is not essential for B cell responses following antigen stimulation in vivo. However, CD4+ T helper function was severely diminished in CD45 exon 6-deficient mice, suggesting that the requirements of CD45 molecules in antigen-specific response and development are different between T and B lymphocytes.

Interleukin (IL)-4 and IL-5 are two cytokines which synergize in the induction of several biological effector functions. They are produced by mouse and human T helper 2 (Th2) and T helper 0 (Th0) cells. Little is known about the regulation of the two cytokines at the single-cell level. Here we show, using a flow cytometric intracellular staining technique, that IL-4 and IL-5 are predominantly produced by different human peripheral CD4+ and CD8+ T cells, whereas interferon (IFN)-gamma and IL-2 are produced by the same cells. In contrast, cloned human Th0 and Th2 cells were able to produce IL-4 and IL-5 simultaneously. The segregation of IL-4 and IL-5 in activated peripheral T cells was found within 72 h of activation upon anti-CD3 or phorbol ester + ionomycin stimulation. The kinetics of IL-4 and IL-5 production were different at the mRNA and the intra- and extracellular protein level, indicating that the cytokines are regulated differently. T cells from three patients with hyper-IgE syndrome did not display a substantial proportion of IL-4/IL-5 double-positive cells. However, simultaneous production could be induced in normal human T cells after prolonged stimulation with a minimum of two restimulation cycles. We conclude that the simultaneous production of IL-4 and IL-5 is a feature of repetitively activated human T cells.

Four monoclonal antibodies (mAb) termed NKTA255, NKTA72, 1F1 and 1B1 were selected on the basis of their ability to inhibit the cytolytic activity of natural killer (NK) cell clones against P815 target cells. These mAb selectively reacted with normal or tumor cells of hematopoietic origin and displayed a cellular distribution similar to that of CD45 or CD11a/CD18 antigens. Immunoprecipitation experiments showed that they reacted with molecules with an apparent molecular mass of 40 kDa under both reducing and nonreducing conditions ("p40" molecules), thus differing from CD45 or CD11a/CD18 antigens as well as from the "inhibitory" receptors for HLA class I molecules (i.e. p58, CD94 and NKB1 molecules). Double-immunofluorescence analysis of peripheral blood mononuclear cells allowed the identification of three distinct populations on the basis of the fluorescence intensity of cells stained with anti-p40 mAb. p40bright cells were homogeneously HLA-DR-positive, p40medium cells were HLA-DR-negative but co-expressed CD56 antigens, while p40dull cells were all CD3+. Anti-p40 mAb strongly inhibited the lysis of K562 target cells, mediated by fresh NK cells, as well as the lysis of P815 target cells by NK or T cell clones. In addition, in redirected killing assays, anti-p40 mAb strongly reduced the anti-CD16 mAb-induced cytolytic activity of NK cell clones. On the contrary, they did not inhibit either the anti-CD3 or anti-T cell receptor mAb-mediated cytolytic activity of T cell clones or the lysis of allogeneic phytohemagglutinin blasts mediated by specific cytolytic T cell clones. The p40-induced inhibition of the NK cytotoxicity required optimal cross-linking, as anti-p40 mAb could inhibit the lysis of Fc gamma receptor (Fc gamma R)-positive but not of Fc gamma R-negative target cells. In addition, (Fab')2 fragments of anti-p40 mAb failed to inhibit the lysis of Fc gamma R-positive target cells. In conclusion, p40 molecules represent a new type of inhibitory surface molecule that appears to play a general regulatory role in the NK-mediated cytolysis.

Human Langerhans cells (LC) express CD45, but clear data about the isoform(s) and their function(s) are lacking. In the present study, double labeling experiments reveal that freshly isolated LC from normal skin are CD45RO+/RA-/RB-. However, after isolation and short-time culture where LC undergo an in vitro maturation resembling that to lymphoid dendritic cells, CD45RB emerges whereas CD45RO expression decreases. This evolution results from dynamic alternative RNA splicing. Addition of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor-alpha to short-time cultures has no significant effect on CD45RB, but both cytokines accelerate the loss of CD45RO. LC isolated from lesional skin of atopic eczema highly express CD45RO and CD45RB. Cross-linking of CD45 on LC isolated from atopic individuals inhibits the calcium mobilization in response to activation via Fc epsilon receptor type I (Fc epsilon RI). Hence, the protein tyrosine phosphatase CD45 from human LC is subjected to a splicing phenomenon related to the differentiation and activation stage of these cells and regulates their Fc epsilon RI-mediated activation.

CD45RA+ cells have been described to be less responsive to CD3/T cell receptor (TcR)-mediated activation than CD45R0+ T cells. To analyze the underlying mechanism of the differential responses we compared CD3/TcR-triggered tyrosine phosphorylation in the two subsets and studied the role of co-stimulatory signals provided either by accessory cells or pharmacologic activation of protein kinase C by phorbol ester. Stimulation of purified CD45RA+ and CD45R0+ T cells with CD3/TcR antibodies induced similar patterns and intensities of tyrosine phosphorylation in the two subsets, but no proliferation. If accessory cells were used as the source of co-stimulatory signals, strong expression of the 55-kDa chain of the interleukin-2 (IL-2) receptor (CD25), significant IL-2 production and vigorous proliferation were observed in CD45R0+ cells, whereas CD45RA+ cells responded weakly. However, when CD3/TcR-mediated triggering was combined with activation of protein kinase C by phorbol ester, CD45RA+ cells responded strongly. These data indicate that the transmembrane signaling capacity of the T cell receptor expressed by CD45RA+ and CD45R0+ cells is similar and, therefore, is presumably not responsible for the differential reactivities of the two subsets. It is more likely that co-stimulatory signals determine whether CD3/TcR-initiated activation results in strong or weak responses.

Relationships between CD45 and p56lck have been suggested by co-immunoprecipitation of both proteins and by dephosphorylation of the p56lck regulatory site, Tyr 505, by CD45 in vitro. We investigated whether the kinase activity of p56lck is modulated in T cells triggered via CD45. We showed that incubation of Jurkat cells with a combination of two anti-CD45 monoclonal antibodies (mAb) (MC5/2 + D3/9) induced an increase in p56lck kinase activity, while a single mAb did not. Under these conditions, p56lck underwent two consecutive waves of activation. This was accompanied by internalization of the kinase and by a time-dependent increased accessibility of CD45 phosphatase at the plasma membrane. Similarly, activation and internalization of p56lck were observed using a combination of anti-CD45 (MC5/2) and anti-CD2 (T11(2)) mAb, suggesting that a functional complex consisting of CD45, CD2 and p56lck was formed upon cell triggering. Taken together, these results suggests that: (i) CD45 participates in the regulation of p56lck kinase activity in vivo and that (ii) CD45 could play a mediator role in the stimulation and endocytosis of p56lck through the CD2 pathway.

We demonstrate that pretreatment of primary CD4+, but not CD8+ T cells with anti-CD45 inhibits activation signals induced through the T cell receptor for antigen (TCR alpha beta). Specifically, anti-TCR alpha beta-mediated tyrosine phosphorylation of phospholipase C-gamma 1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca2+ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti-CD45 in CD8+ T cells. Perturbation of TCR alpha beta signalling in CD4+ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti-CD45 ablates TCR alpha beta signalling, anti-CD3 epsilon-mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCR alpha beta to second messenger-generating pathways is under distinct physical and/or functional constraints in primary CD4+ and CD8+ T cells.

In rheumatoid arthritis and other inflammatory diseases we and others have found that gamma delta T cells express activation antigens, suggesting that they are involved in the pathogenesis of these disorders. In this study we have stimulated peripheral blood mononuclear cells from normal donors with recombinant interleukin-2 (rIL-2) to see whether such a stimulus alone could activate gamma delta T cells. Short-term exposure (24-96 h) to rIL-2 selectively stimulated the gamma delta but not the alpha beta T cells to express activation antigens (CD69, CD25 and HLA-DR). Long-term culture (2 weeks) in rIL-2-containing medium caused a selective increase in the proportion of the gamma delta T cells and a corresponding reduction of the fraction of alpha beta T cells. Limiting dilution analysis revealed that approximately 1/60 of the gamma delta T cells responded to IL-2 in contrast to only 1/250 of the alpha beta T cells. Comparison of the expression of the IL-2 receptor (IL-2R) alpha and beta chains showed that there was a similar expression of the alpha chain on gamma delta and alpha beta T cells whereas the relative density of the beta chain was more than twice as high on gamma delta T cells. Both the IL-2-induced proliferation of gamma delta T cells and the expression of activation antigens on these cells could be inhibited by an anti-IL-2R beta monoclonal antibody (mAb) but not by an anti-IL-2R alpha mAb. Expression of CD69 on gamma delta T cells was dependent neither on the presence of B cells, monocytes, nor alpha beta T cells. Finally, we found that the IL-2-induced expression of CD69 was inhibited by activation of cAMP-dependent protein kinase and by inhibition of the Src-family of the tyrosine protein kinase, but not by inhibition of protein kinase C or by activation of the CD45 associated tyrosine phosphatase. The ability of gamma delta T cells to be activated by IL-2 is a feature which they have in common with natural killer cells. Moreover, it may be possible that the expression of activation antigens on gamma delta T cells in inflammatory diseases is an epiphenomenon secondary to IL-2 produced by activated alpha beta T cells.

T cell activation by triggering the T cell receptor (TcR)-CD3 complex leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins. To date, there has been no direct evidence on the identity of the tyrosine kinase activity implicated in this signaling pathway. In this study, we demonstrate that activation of human T cells with anti-CD3 monoclonal antibody increases tyrosine kinase activity of p56lck. This extends our previous findings which demonstrated the involvement of p56lck kinase activity in the CD2 signal transduction pathway. The results from peripheral blood lymphocytes and Jurkat cell line showed in both cases an early and transient change in the specific activity of p56lck, followed by a shift to a higher apparent molecular mass. Therefore, to test directly the role of TcR-CD3 in CD2-induced activation of p56lck, we utilized mutant variants of the Jurkat cell line lacking in cell surface TcR-CD3. We found that cell surface expression of TcR-CD3 is not required for the activation of p56lck via CD2 but is necessary for the appearance of the reduced-electrophoretic-mobility form of p56lck observed after CD2 triggering. By isolating CD45- mutants from Jurkat cells, we observed that surface expression of the tyrosine phosphatase CD45 is required in order to increase p56lck activity following CD2 stimulation, while CD4-induced activation of the kinase remained unchanged. These data provide evidence for a specific functional linkage between CD2 and p56lck, in which CD45 may play an essential role.